The Epidemiology Branch is conducting a number of birth defect studies in collaboration with the Health Research Board and Trinity College, Dublin, Ireland. The main objective of these studies is to determine the relationship between folate and birth defects. The birth defects studied to date are neural tube defects (NTDs), oral clefts, congenital heart defects, Down syndrome and omphalocele. These studies focus on biochemical factors in the area of folate metabolism, and on genetic mutations in folate related genes associated with birth defects. In the past we have shown that elevated homocysteine is a risk factor for NTDs, that a mutation in the methylenetetrahydrofolate reductase (MTHFR) gene 677C->T is a risk factor for NTDs, and that a small dose of folic acid (100-200 micrograms) can raise red cell folate to levels that can prevent a fifth to almost a half of NTDs. We have shown that methylenetetrahydrofolate reductase (MTHFD), an important gene in the production of purine and pyrimidine for DNA synthesis, is a risk factor for NTDs. A report from another institution claimed that 75% of women who were mothers of children with neural tube defects (NTDs) had auto-antibodies to the folate receptor vs. only 10% of control mothers. We attempted to confirm these findings in our large Irish population. Our results showed that mothers of NTD children were no more likely to have folate-receptor autoantibodies than controls, or than women who had never been pregnant. This is an important result because it had been postulated that failure of folate to bind with its receptor could be a major causal mechanism for NTDs. This result has been published in the New England Journal of Medicine. For many years investigators have found that women delivering babies with NTDs had lower vitamin B12 levels than women delivering unaffected babies. Many investigators believe that this in not simply a case of women at risk having diets poor in both vitamins, but that B12 is an independent risk factor. We recently published data addressing this question and the follow up question: if B12 is a risk factor, how high must a woman's B12 level be to avoid the risk? Our data show that B12 is a significant risk factor after controlling for the effect of folate levels. Moreover, maternal B12 levels in the deficient or borderline range substantially increased the NTD risk. Once levels reached the normal range, there was no additional benefit from having higher B12. We have expanded our genetic investigation greatly by using Illumina technology to examine 1536 single nucleotide polymorphisms (SNPs) from candidate genes to identify other genes associated with NTDs. Cases, their mothers, their fathers (triads), and unaffected controls have been genotyped. The Illumina run has been completed and the SNPs that showed the strongest association with NTDs have been identified. A second, confirmatory group of triads and unaffected controls has been genotyped for these and related SNPs. The data have now been editing and are being analyzed. As part of this project, Dr. Troendle has developed a method of testing for genetic association with constrained models using triads. As part of our ongoing investigation of genetic factors related to NTDs, we examined the promoter of MTHFD1, a gene previously shown by us to be a maternal risk factor for NTDs. We found that a common SNP was not an independent risk factor but was associated with increased risk in combination with the previously reported risk factor SNP, R653Q (rs2236225). This study also provided data on how MTHFD1 is regulated. Because of the known association between maternal obesity and NTD risk (first reported by Waller et al. in a previous study done by us on NTDs), we examined the uncoupling protein UCP2 polymorphisms as possible NTD risks. UCP2 has been postulated to have a role in obesity. Our investigation, however, did not confirm a published study indicating that NTD risk was increased in those with common variants in the UCP2 gene.